Nucleic acid-based therapies useful in vaccination and in delivery of polypeptides and gene replacement have made great advancements in recent years (for a brief review, see, e.g., Hewson (2000) Mol. Med Today 6:28–35). However the search for vehicles that provide for efficient delivery and expression of a sequence of interest without inducing undesirable side effects continues. One particular problem in this context has been in the use of viral vectors. Viral vectors exploit the natural ability of viruses to delivery genetic material to cells and use the host cell machinery to provide for expression of the encoded gene product and/or incorporation of the sequence into the host genome (as either a integrated or episomal entity). However, the transfer of the therapeutic recombinant gene to host cells not only results in expression of the gene product of interest, but may also result in the synthesis of viral proteins. Cells that express these viral proteins are recognized and killed by cytotoxic T lymphocytes, which in turn eradicate the transduced cells, promote release of cytokines, and mediate inflammation through release of cytokines. Other vectors that provide for expression of the encoded gene products in the cytoplasm of the host cell meet with the same or similar problems.
Vectors based upon picornaviral genomes have been of interest due to the widespread success of live vaccines in the now nearly world-wide eradication of poliovirus. Picornaviruses are extremely prevalent and successful viruses, replicating abundantly in organisms ranging from insects to humans. Picornaviruses include polioviruses, rhinoviruses, coxsackieviruses, and echoviruses. Picornaviruses are non-enveloped viruses that encode no known glycosylated or transmembrane proteins. However, poliovirus, the most extensively studied picornavirus, encodes at least three non-structural proteins that drastically affect host intracellular membrane structure and function. Specifically, poliovirus protein 2C induces membrane vesiculation while proteins 2B and 3A are each sufficient to inhibit protein traffic through the host secretory pathway (Aldabe et al. (1995) Biochem. Biophys. Res. Commun. 206:64–76; Bienz et al. (1987) Virology 160:220–226; Cho et al. (1994) Virology 202:129–145, Dales et al. (19 Virology 26: 379–389; Doedens et al. (1995) Embo J 14:894–907; Doedens et al. (1997) J. Virol. 71:9054–9064; Schlegel et al. (1996) J. Virol. 70:6576–65887). In isolation, protein 3A interacts with endoplasmic reticulum (ER) membranes to inhibit protein transport from the ER to the Golgi apparatus (Doedens et al. (1995) Embo J 14:894–9; Doedens et al. (1997) J. Virol. 71:9054–9064).
Until the work described herein, the role of inhibition of ER-to-Golgi traffic during viral infection was as yet unknown One possibility was that inhibition of protein secretion results from construction of a structural scaffold for the viral-RNA replication complex. Poliovirus RNA replication occurs on the cytoplasmic surface of double-membraned vesicles that proliferate in virally infected cells (Bienz et al. (1987) Virology 160:220–226; Dales et al. (1965) Virology 26:379–389; Schlegel et al. (1996) J. Virol. 70:6576–6588). All of the viral proteins required for RNA replication (2B, 2BC, 3A, 3AB, 3CD, and 3D) are physically associated with these vesicles in infected cells (Bienz et al. (1992) J. Virol. 66:2740–2747; Bienz et al. (1994) Arch. Virol. Suppl. 9:147–157). Another possibility was that inhibition of protein secretion is not required for RNA replication complex function, but is a virulence factor that enhances viral infection in tissues and animals. Consistent with the second hypothesis, the functions of 3A in viral RNA replication and in inhibiting protein secretion can be genetically separated Specifically, a mutant poliovirus, 3A-2 (Bernstein et al. (1988) J. Virol. 62:2922–2928), contains a mutation in the 3A protein that renders it much less efficient at blocking ER-to-Golgi traffic (Doedens et al (1997) J. Virol. 71:9054–906441), yet only causes a slight growth defect. However, this previous line of investigation did not definitively identify the role of 3A proteins, leaving both possibilities open. For example, other groups reported that recombinat, intact poliovirus genomes constructed to express an exogenous antigen induced protective CTL mediated immunity (Mandi et al. (1998) Proc. Natl. Acad. Sci. USA 95:8216–8221). However, the amount of this CTL response was not quantified, and the experimental system was chosen so that even low levels of CTL response would be protective. Therefore, the role of picornaviral protein 3A in viral pathogenesis and in manipulation of the host cellular machinery is not understood.
To date, strategies using the poliovirus genome as a vectors for expression of other proteins or peptides have included live recombinant viruses that could replicate and spread within infected cells or organisms (Alexander et al. (1994) Proc. Natl. Acad. Sci. USA 91:1406–1410; Andino et al. (1994) Science 265:1448–1451; Burke et al. (1988) Nature 332:81–82; Mandl et al. (1998) Proc. Natl. Acad. Sci. USA 95:8216–8221; Mattion et al. (1994) J. Virol. 68:3925–3933) as well as RNA “replicons” that can only undergo one infectious cycle in target cells and tissues (Ansardi et al. (1994) 54:6359–6364; Poon et al. (1999) EMBO J. 18: 555–564). Advantages of poliovirus-based vectors include their decades of use in humans, their ease of administration, and their ability to induce protective antibody responses. The role of viral proteins in picornaviral infections would provide not only a better understanding of viral pathogenesis, but also insight as to how to develop recombinant viral vectors that provide for the desired immune response, conferring the ability to modulate (e.g., increase or decrese) inflammatory and CTL response. The present invention addresses these issues.